Human Cancer Biology Tumor-Driven Paracrine Platelet-Derived Growth Factor Receptor A Signaling Is a Key Determinant of Stromal Cell Recruitment inaModelofHumanLungCarcinoma Max L. Tejada,1Lanlan Yu,1Jianying Dong,1Kenneth Jung,3 Gloria Meng,4 Franklin V. Peale,2 Gretchen D. Frantz,2 Linda Hall,2 XiaoHuan Liang,1Hans-Peter Gerber,1and Napoleone Ferrara1 Abstract Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRa ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRa was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRa-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRa in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors. It has become apparent that the microenvironment in which markersconsistentwiththosefoundatthesitesofnormal tumor cells develop profoundly influences many steps of tumor wound healing (3). progression. The tumor microenvironment consists of a stroma, In the normal wound repair process, fibroblasts are recruited which is composed of immune and inflammatory cells, from the surrounding tissue. However, cancer cells are capable endothelial cells and fibroblasts, a variety of growth factors of recruiting fibroblasts from the surrounding environment and and cytokines, and an insoluble extracellular matrix. In various inducing their activation to the myofibroblast phenotype experimental tumor models, each of the stromal cell types has directly (3). In addition to their presence in the tumor mass, been shown to influence the efficiency of tumor formation, the myofibroblasts are often found at the invasive fronts of a rate of tumor growth, the extent of invasiveness, and the ability number of solid tumors, where they have been shown to affect of the tumor cells to metastasize (1–3). the growth, invasion, and metastasis of the tumors through Much attention has been focused on the role of their increased synthesis of extracellular matrix components endothelial cells in tumor angiogenesis; however, there is and the increased production of proteases, growth, and accumulating evidence that in many solid tumor types, the angiogenic factors (4). influence of the microenvironment is mediated in large part The growth of solid tumors requires the formation of new by paracrine signaling between the tumor epithelium and blood vessels through angiogenesis, the sprouting of new blood neighboring stromal fibroblasts (4). Fibroblasts are the vessels from preexisting ones, although more recent evidence major constituent of the stroma in many solid tumors, and indicates that recruitment of bone marrow–derived angiogenic in some cases, the fibroblast stroma comprises >90% of the cells contributes to the development of a neovascular supply tumor mass. These tumor-associated fibroblasts often exist in (5, 6). Although a number of angiogenic factors have been a constitutively activated state and often exhibit biological identified, vascular endothelial growth factor A (VEGF-A) is probably the most ubiquitous, and much experimental evidence implicates this factor in the regulation of both physiologic Authors’ Affiliations: Departments of 1Molecular Oncology, 2Pathology, and pathologic angiogenesis (7, 8). Administration of an anti- 3Bioinformatics and Assay, and 4Automation Technology, Genentech, Inc., South VEGF-A humanized monoclonal antibody (mAb; bevacizumab) San Francisco, California in combination with chemotherapy results in increased survival Received 8/12/05; revised 12/22/05; accepted 2/17/06. relative to chemotherapy alone in patients with previously The costs of publication of this article were defrayed in part by the payment of page untreated metastatic colorectal cancer (9). In several experimen- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tal systems, inactivation of the VEGF-A gene indicates that Note: Supplementary data for this article are available at Clinical Cancer Research tumor-derived VEGF is important for tumor angiogenesis and Online (http://clincancerres.aacrjournals.org/). growth (10, 11). However, other studies have detected significant Requests for reprints: Napoleone Ferrara, Department of Molecular Oncology, levels of VEGF in the stromal compartment, suggesting a Genentech, Inc., 1DNA Way, South San Francisco, CA 94080. Phone: 650-225- 2968; Fax: 650-225-6443; E-mail: [email protected]. significant role for stromal fibroblast-derived VEGF (12, 13). F 2006 American Association for Cancer Research. Members of the platelet-derived growth factor (PDGF) family doi:10.1158/1078-0432.CCR-05-1770 have been shown to be potent mitogens and chemoattractants Clin Cancer Res 2006;12(9) May 1, 2006 2676 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. PDGFRa Signaling and Human Lung Carcinoma j for a wide variety of cell types of mesenchymal origin (14). In were incubated at 37 C in an atmosphere of 95% air/5% CO2. The both human and mouse, the PDGF family consists of four human embryonic kidney 293cell line (American Type Culture members (PDGFA, PDGFB, PDGFC, and PDGFD), which exert Collection), used to generate the adenovirus used in these experiments, their biological effects by binding to two receptor tyrosine was typically grown using high-glucose DMEM supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin/streptomycin, 2 mmol/L kinases (PDGFRa and PDGFRh). PDGFAA, PDGFAB, PDGFBB, L-glutamine, and 1 Ag/mL Fungizone (Invitrogen). a and PDGFCC dimers bind to PDGFR with high affinity, Generation and fractionation of conditioned medium. Calu-6 cells h whereas PDGFBB and PDGFDD dimers bind PDGFR prefer- were allowed to grow to confluency in the presence of growth medium entially. PDGF signaling is critical for proper embryonic on gelatin-coated 500-cm2 dishes. The medium was then replaced with development, whereas in the adult, it plays a role in wound serum-free DMEM/Ham’s F12 medium, and the cells were incubated for healing and in the control of interstitial fluid pressure (14). an additional 72 hours, at which time the media (total of 6 liters) was Aberrant PDGF signaling is a hallmark of a number of solid harvested and concentrated 5-fold using a Filtron Ultrasette tangential tumors, and studies of breast, lung, and colon cancers have flow device with 10K membrane (Filtron Technology Corp., North- shown a tight correlation between deregulated paracrine PDGF borough, MA). All fractionation procedures described were done using signaling and cancer progression (15–17). Furthermore, in an AKTA Explorer chromatography system (GE Healthcare Bio-Sciences gliomas, fibrosarcomas, and osteosarcomas, coexpression of the Corp., Piscataway, NJ). Cation-exchange column chromatography. The concentrated medium PDGF ligands and their cognate receptors by the tumor cells was dialyzed against 25 mmol/L sodium phosphate (pH 6) and applied leads to an autocrine mechanism that drives carcinogenesis on a 5-mL HiTrapS Sepharose column (Amersham Pharmacia Biotech), (18–20). Unlike the other members of its family, PDGFAA which had been equilibrated with the same buffer. After a low salt exhibits a weak transforming activity, and thus far, most of the wash, the column was eluted with 1 mol/L NaCl. studies have focused on its role in embryonic development (21). Size exclusion column chromatography. Bioactive 1 mol/L NaCl Many studies have focused upon the role of tumor-derived fractions generated using cation-exchange chromatography were factors, such as PDGFB, basic fibroblast growth factor, and pooled, and 5-mL aliquots were loaded onto a TSK3000 column transforming growth factor h-1 in the induction of the myofibro- (21.5 Â 30 cm; Tosoh Biosep LC, Montgomeryville, PA) that had been blast phenotype and the angiogenic process (22–24). However, equilibrated in 20 mmol/L Tris (pH 7.5), 2 mol/L NaCl, and 0.02% attempts to identify bona fide tumor-derived stromal fibroblast Tween 20. The flow rate was 3mL/min, 3-mLfractions were collected, and aliquots were tested for bioactivity. recruitment factors are rare, despite the fact that intuitively, this Reverse-phase column chromatography. Bioactive fractions generated precedes fibroblast activation (25–27). In addition, using a using size exclusion chromatography were pooled, diluted 5-fold in dominant-negative strategy, Shao et al. showed that the expression water containing 0.1% trifluoroacetic acid and loaded onto a C4 4000A of several PDGF family members was responsible for a desmo- column (4.6 Â 100 mm). The column was eluted using a linear gradient plastic response in mammary gland tumor xenografts (28). of 15% to 50% acetonitrile (120 minutes) in 0.1% (v/v) trifluoroacetic Recently,